Figure 2.

Nedd8 is required for p27 ubiquitination. (A) Ubiquitination of p27 requires a FI component. In vitro-translated [³⁵S]His₆-p27 was incubated in the absence (lane 1) or presence of FII (lanes 2–6). FII containing reactions were further supplemented by the addition of buffer alone (lane 2), a low molecular weight gel filtration fraction of FI (lane 3), or 0.29 μM Nedd8 (lane 4). Substitution of ubiquitin with Ub-K48R results in the formation of discrete low molecular weight p27-ubiquitin conjugates in the presence (lane 5) but not the absence of recombinant Nedd8 (lane 6). (B) Ubiquitination of p27 by FII requires both Nedd8 and the Nedd8-conjugating enzyme, Nce1. GST-p27 was incubated with FII alone (lane 1) or in the presence of 1 μM Nce1 and/or 0.29 μM recombinant Nedd8 as indicated (lanes 2–4). (C) Prior phosphorylation is required for p27 ubiquitination. Reactions were performed by using S100 extract as a source of ubiquitinating enzymes, and either in vitro-translated [³⁵S]His₆-p27 or bacterially expressed GST-p27 as substrates. Ubquitination of [³⁵S]His₆-p27 (lanes 1–3) was carried out in the absence (lane 1) or presence of 2 mM ATP (lane 2) or 2 mM AMP-PNP (lane 3). In lanes 4–6, GST-p27 (F62/64A) was prephosphorylated with cyclin E/CDK2 using [γ-³²P]ATP and repurified. Reactions containing [³²P]-GST p27 were conducted in the absence (lane 4) or presence of 2 mM ATP (lane 5) or 2 mM AMP-PNP (lane 6). (D) Ubiquitination of prephosphorylated [³²P]-GST-p27 by FII requires both Nedd8 and Nce1 (lanes 1–3).